Glucose isomerase (E.C.5.3.1.5) or xylose isomerase or D-xylose ketol isomerase is a key enzyme in the pentose phosphate pathway, catalyzing the conversion of D-xylose to D-xylulose. The isomerase also converts D-glucose to fructose and therefore is one of the most important enzymes in the food and beverage industry for the manufacture of fructose syrup (Kaneko et al., Biosci Biotechnol Biochem. 2000, 64: 940-947). The equilibrium of the isomerization of D-glucose to fructose is primarily dictated by the temperature of the reaction. The higher the temperature, the more the fructose in the final mixture of fructose and glucose. At present the commercial glucose isomerases come mainly from Actinoplanes missouriensis, Bacillus coagulans, Streptomyces rubiginosus and Streptomyces murinus, and are not stable at temperature above 65° C. Consequently, the current commercial isomerization is restricted to operate at around 60° C. and the products normally contain no more than 44% of fructose. The HFCS-55 (high fructose corn syrup containing 55% fructose) used in beverage and other industries is usually obtained by expensive chromatographic enrichment.
The scientists around the world have been working on the identification and protein engineering of thermostable and highly active glucose isomerases from thermophilic bacteria. J. G. Zeikus and his collaborators have isolated and studied thermostable glucose isomerases from thermophilic bacteria Thermoanaerobacterium saccharolyticum and Thermotoga neapolitana (Lee et al., Journal of General Microbiology, 139:1227-1234, 1993; Vieille et al., Methods Enzymology, 330:215-24, 2001; Lee et al., Journal of General Microbiology, 139:1241-1243, 1993; Scriprapundh et al., Protein Engineering, 13:259-265, 2000); Scriprapundh et al., Protein Engineering, 16:683-690, 2003; Zeikus et al., U.S. Pat. No. 5,656,497)). Nevertheless, the thermostability and the activity of these and other thermostable glucose isomerases have not yet met the requirements for industry applications. Thus, a glucose isomerases having improved activity or improved activity and thermostability is still desired for the industrial application.